GENE REGULATION BY TRASCRIPTION ANTITERMINATION

描述：本项目继续对基础知识的阐述 控制转录延长和终止的机制 细菌和噬菌体。 关于延伸率控制的两个大的工作领域，寻求实现几个 基因和生化相结合的方法的主要目标是 建议。第一个研究领域，这笔赠款的新方向， 重点介绍了两种新药的作用和作用机制。 已发现的大肠杆菌、GreA和Greb的伸长因子，以及 最近发现的合作者(或抑制者)基因的功能， 格雷克。GREA和GREB是细胞生长所必需的 温度。GREC突变绕过了这一要求。在体外，GREA 和Greb促进RNA聚合酶(RNAP)对转录的切割，并通过 这一活动的优点，可能在转录中扮演多种角色： 抑制流产的启动(并因此刺激 有效伸长和新的起始)，从伸长停止中解脱， 和增强转录保真度。这是否在体内是真的 是被确定的，由grea，greb调控的关键靶基因 和grec还有待鉴定，涉及的分子机制 每个监管事件都将被剖析。 第二个领域扩展了正在进行的以噬菌体L N为中心的研究 基因蛋白是一种原型的抗终止剂，它与独特的 结构，捕获RNAP，并与 细胞蛋白质，将RNAP转化为抗终止 州政府。需要检验的关键假设是N蛋白和 其RNA靶标通过掩蔽或 修饰聚合酶中对终止和 暂停。建议确定RNAP的亚基，并本地化 联系N及其合作者的位点：顺式作用的RNA 茎环结构与寄主因子NusA、NusG和核糖体 蛋白质S10。还提出了对区域进行局部化和解剖的方法 在识别A、RNAP和NUSA的N个中，试图推导出基本的 RNA-蛋白质和蛋白质-蛋白质相互作用的原理，以及 说明潜在的变构修饰。它进一步提出了 为了揭示N和另一个宿主因子NusG， 通过暂停位置加速RNAP并稳定延伸率 构象，这一现象可能是 终止抑制。额外的正负调制器 可能在噬菌体裂解-溶源决定中起作用的N 发展是孤立的。对一种细胞的有力的基因搜索 N和由类N反终止激活的靶基因的同源物 将采取的机制。有人断言，这些研究应该 提供对RNA聚合酶关系的基本见解 具有功能的结构和基因调控机制。

DESCRIPTION: This project continues the elucidation of fundamental mechanisms that control transcription elongation and termination in bacteria and bacteriophages. Two broad areas of work on elongation control, seeking to achieve several major objectives by combined genetic and biochemical approaches, are proposed. The first area of study, a new direction for this grant, focuses on the function and the mechanism of action of two recently discovered elongation factors of E. coli, GreA and GreB, and the function of a recently discovered collaborator (or suppressor) gene, greC. GreA and GreB are essential for cell growth at elevated temperatures. GreC mutations bypass this requirement. In vitro, GreA and GreB promote transcript cleavage by RNA polymerase (RNAP), and, by virtue of this activity, may play multiple roles in transcription: suppression of abortive initiation (and, consequently, stimulation of productive elongation and new initiation), relief from elongation arrest, and enhancement of transcription fidelity. Whether this is true in vivo is to be determined, key target genes that are regulated by GreA, GreB and GreC are to be identified, and the molecular mechanisms involved in each regulatory event are to be dissected. The second area extends ongoing studies centering around the phage l N gene protein, the archetype antiterminator that binds to a unique structure in the nascent mRNA, captures RNAP and, in conjunction with cellular proteins, transforms that RNAP to a termination-resistant state. The critical hypothesis to be tested is that the N protein and its RNA target lock RNAP into the stable elongation state by masking or modifying sites in polymerase that are critical for termination and pausing. It is proposed to identify the subunits of RNAP, and localize the sites that contact N and its collaborators: the cis-acting RNA stem-loop structure and the host factors NusA, NusG and the ribosomal protein S10. It is also proposed to localize and dissect the domains of N that recognize A, RNAP and NusA, attempt to deduce the basic principles of RNA-protein and protein-protein interactions, and illuminate the potential allosteric modifications. It further proposed to uncover the mechanisms by which N and another host factor, NusG, accelerate RNAP through pause sites and stabilize the elongation conformation, phenomena that are likely to be quintessential for termination suppression. Additional positive and negative modulators of N that might play a role in lysis-lysogeny decisions of phage development are to be isolated. A vigorous genetic hunt for a cellular homologue of N and target genes activated by an N-like antitermination mechanism will be undertaken. It is asserted that these studies should provide fundamental insights into the relationships of RNA polymerase structures with functions, and mechanisms of gene regulation.